A clinically compatible drug‐screening platform based on organotypic cultures identifies vulnerabilities to prevent and treat brain metastasis

Abstract We report a medium‐throughput drug‐screening platform (METPlatform) based on organotypic cultures that allows to evaluate inhibitors against metastases growing in situ. By applying this approach to the unmet clinical need of brain metastasis, we identified several vulnerabilities. Among them, a blood–brain barrier permeable HSP90 inhibitor showed high potency against mouse and human brain metastases at clinically relevant stages of the disease, including a novel model of local relapse after neurosurgery. Furthermore, in situ proteomic analysis applied to metastases treated with the chaperone inhibitor uncovered a novel molecular program in brain metastasis, which includes biomarkers of poor prognosis and actionable mechanisms of resistance. Our work validates METPlatform as a potent resource for metastasis research integrating drug‐screening and unbiased omic approaches that is compatible with human samples. Thus, this clinically relevant strategy is aimed to personalize the management of metastatic disease in the brain and elsewhere.

. Inhibition of HSP90 is effective to treat established brain metastasis.
A Representative images showing HSP70 levels in brain metastases (generated by intracardiac inoculation of H2030-BrM) found at endpoint of vehicle and DEBIO-0932-treated animals. Scale bar: 75 µm. B Quantification of HSP70 levels shown in (A) in arbitrary fluorescent units (A.F.U.). Values are shown in box-and-whisker plots where each dot is a metastatic lesion and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles, and the whiskers go from the minimum to the maximum value (n = 6-12 metastatic lesions from 3 to 6 brains per condition). P value was calculated using two-tailed t-test. C, D HSP90AA1 (C) and HSPB2 (D) expression levels obtained by qRT-PCR of H2030-BrM brain metastases obtained at endpoint of vehicle and DEBIO-0932-treated animals. Values are shown in box-and-whisker plots where every dot represents a different animal and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles, and the whiskers go from the minimum to the maximum value (n = 4 mice per experimental condition). P value was calculated using two-tailed t-test. E Representative images of HSP90 + non-cancer cell compartments including the medial habenula and the Iba1 + microglia/macrophages in the metastasis-associated microenvironment from vehicle and DEBIO-0932-treated brains at the endpoint of the experiment ( Fig 4A). Hematoxylin eosin staining of three organs from vehicle and DEBIO-0932-treated mice at experimental endpoint. (n = 3 mice per experimental condition were evaluated for each organ). Scale bar: 50 µm. L Kaplan-Meier curve comparing overall survival of vehicle and DEBIO-0932-treated mice following the schedule depicted in Fig 4A. (n = 9 mice treated with vehicle and n = 10 mice treated with DEBIO-0932). P value was calculated using log-rank (Mantel-Cox) test. The arrow indicates when the treatment was initiated. M Schema of experimental design. The brain metastatic melanoma cell line B16/F10-BrM was intracranially injected to generate an established tumor so the treatment could start 3 days post-injection. N Representative image of an established tumor 3 days post-injection. The interface between the metastasis and the associated microenvironment is well-defined. Scale bar: 50 µm (low magnification); 25 µm (high magnification). O Representative images of slices with the brain tumor at the end of the experiment. BB: Bisbenzamide. Scale bar: 1 mm. P Quantification of the tumor area at experimental endpoint. Values are shown in box-and-whisker plots where every dot represents a different brain and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value (n = 6 mice per experimental condition). P value was calculated using two-tailed t-test. Q Representative images of human brain metastases from which BrM-PDOC were generated and evaluated as responders ( Fig 4H) that showed no correlation with HSP90-dependent oncogenic drivers ALK and ROS1. Scale bar: 50 µm. R Representative images of human brain metastases from which BrM-PDOC were generated and evaluated as responders ( Fig 4H) that showed positive correlation with HSP90-dependent oncogenic drivers HER2 and BRAF. Scale bar: 50 µm. Targeting sequencing of the EGFR locus of a lung cancer brain metastasis patient showing a deletion in exon 19 is also shown. S Pie chart showing the distribution of the ten BrM-PDOCs with oncogenic drivers sensitive to HSP90 inhibition (Non-HSP90 client: n = 4; EGFR mutant lung cancer: n = 2; HER2 + breast cancer: n = 3; BRAF mutant melanoma: n = 1). vehicle and n = 9 DEBIO-0932-treated mice, 2 independent experiments). P value was calculated using two-tailed t-test (P values: *P < 0.05, ***P < 0.001). D Representative images of brains and thorax from vehicle and DEBIO-0932-treated mice at the endpoint of the experiment. E, F Quantification of ex vivo BLI of brains (E) and thoracic regions (F) at the endpoint of the experiment. Values are shown in box-and-whisker plots where every dot represents a different animal and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles, and the whiskers go from the minimum to the maximum value (n = 9 vehicle and n = 9 DEBIO-0932-treated mice, 2 independent experiments). P value was calculated using two-tailed t-test. G Kaplan-Meier curve comparing overall survival of vehicle and DEBIO-0932-treated mice starting 7 days post-intracardiac injection (n = 10 mice treated with vehicle and n = 11 mice treated with DEBIO-0932). P value was calculated using log-rank (Mantel-Cox) test. The arrow indicates when the treatment was initiated. H Schema showing the individualized therapy of mice receiving surgery + DEBIO-0932 during the treatment period starting 3 days after surgery. Each row represents a mouse receiving DEBIO-0932 (green) o not (white) (n = 11 surgery + DEBIO-0932-treated mice, 2 independent experiments). Gray squares indicate decease of the corresponding animal. I Graph showing survival of mice treated with surgery + vehicle or surgery + DEBIO-0932. The graph represents each mouse with a bar only if the survival is above the median of the group receiving surgery + vehicle (60.5 days) (n = 8 surgery + vehicle and n = 11 surgery + DEBIO-0932-treated mice, 2 independent experiments). P value was calculated using two-tailed t-test.
A Representative images showing RPLP1 levels in brain metastases (generated by intracardiac inoculation of H2030-BrM) found at endpoint of vehicle and DEBIO-0932-treated animals. This result was reproduced in two independent staining with different brains. Scale bars: 50 µm. B Quantification of RPLP1 and AHR levels shown in (Figs 6E and EV4A) in arbitrary fluorescent units (A.F.U.). Values are shown in box-and-whisker plots where each dot is a metastatic lesion and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles, and the whiskers go from the minimum to the maximum value (n = 8-16 metastatic lesions from 2 to 4 brains per condition, two independent staining with different brains were performed). P value was calculated using two-tailed t-test. C Quantification of percentage of nuclear DDA1 + BB + cells shown in (Fig 6E). Values are shown in box-and-whisker plots where each dot is a metastatic lesion, and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles, and the whiskers go from the minimum to the maximum value (n = 16 metastatic lesions from 4 brains per condition, 2 independent staining with different brains were performed). P value was calculated using two-tailed ttest. D Representative images showing p-ERK levels in organotypic cultures from (Fig 6A). This result was reproduced in three independent staining with organotypic cultures from different mice. Scale bar: 20 µm. ◀ Figure EV5. METPlatform as a clinically compatible "avatar".
A Pie chart categorizing the 17 GB-PDOC treated with radiation (Rx) and temozolomide (TMZ) into responders (R) and non-responders (NR) independently of the dose of TMZ given (gray). Red area is labeling the single PDOC with a non-homogeneous response to the two different doses of TMZ (D). B-D Quantification of the impact of Rx + TMZ on the proliferation of cancer cells from GB-PDOC. Values are normalized to the respective control without treatment.
Each dot represents an organotypic culture from each GB-PDOC. GB-PDOC was classified as responder when the mean (line in each dot cluster) is below 40%. P value was calculated using two-tailed t-test. E Representative images of a responder GB-PDOC stained with a DNA-damage marker, c-H2AX (red). BB: bisbenzamide. Scale bar: 25 µm. F Quantification of the number of c-H2AX + cancer cells in the GB-PDOC shown in (E). Each dot represents and individual organotypic culture where the mean number of cells positive for the DNA-damage marker was measured. The dotted line represents the mean AE s.e.m. (n = 3, uncultured organotypic slices from the tumor; n = 4 GB-PDOC treated with DMSO (1%); n = 5 GB-PDOC treated with Rx + TMZ (25 lM); n = 5 GB-PDOC treated with Rx + TMZ (250 lM)). P value was calculated using two-tailed t-test. G Quantification of LDH levels in the conditioned media of organotypic slices cultured during 3 and 7 days relative to a lysate of the same GB-PDOC. Values are shown as mean + s.e.m. (n = 3 organotypic cultures per experimental condition, each graph correspond to an individual GB-PDOC). LDH levels at 7 days measure the accumulation of the enzyme in the media from day 3 on. H Post hoc quantification of the ratio between the number of months with stable disease in each patient (at cut off September 2021) and the corresponding % of proliferation after GB-PDOC treatment in METPlatform. The dotted line indicates the median. Each dot corresponds to a patient. Green dots: patient with stable disease; white dots: patients with progressive disease. The red line labels those patients with a reduction above 40% in cancer cell proliferation in the corresponding GB-PDOC.